Synthesis and biological evaluation of alkoxycoumarins as novel nematicidal constituents

Article abstract of DOI:10.1016/j.bmcl.2008.08.102

We synthesized all of the monomethoxycoumarins, 5-alkoxycoumarins and their derivatives, and investigated their nematicidal activity against the phytopathogenic nematode, Bursaphelenchus xylophilus. Among the compounds, 5-ethoxycoumarin showed the highest nematicidal activity. Furthermore, 5-ethoxycoumarin was comparatively harmless against both the brine shrimps, Artemia salina, and the Japanese killifish, Oryzias latipes.

Full text of DOI:10.1016/j.bmcl.2008.08.102

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5614–5617
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of alkoxycoumarins as novel
nematicidal constituents
a
a
a
a
Kazuto Takaishi a,b, , Minoru Izumi , Naomichi Baba , Kazuyoshi Kawazu , Shuhei Nakajima
*
a Laboratory of Applied Natural Products Chemistry, The Graduate School of Natural Science and Technology, Okayama University, 1-1-1 Tsushimanaka,
Okayama, Okayama 700-8530, Japan
b Supramolecular Science Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2008
Revised 26 August 2008
Accepted 28 August 2008
Available online 31 August 2008
We synthesized all of the monomethoxycoumarins, 5-alkoxycoumarins and their derivatives, and inves-
tigated their nematicidal activity against the phytopathogenic nematode, Bursaphelenchus xylophilus.
Among the compounds, 5-ethoxycoumarin showed the highest nematicidal activity. Furthermore, 5-eth-
oxycoumarin was comparatively harmless against both the brine shrimps, Artemia salina, and the Japa-
nese killifish, Oryzias latipes.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
5-Alkoxycoumarins
Nematicide
Bursaphelencus xylophilus
Artemia salina
Oryzias latipes
In Japan, China and the surrounding countries, a serious wilting
disease is epidemic in pine wood.1 This disease is caused by a nem-
atode, Bursaphelencus xylophilus,2 and the nematode is transmitted
by a longhorn beetle, Monochamus alternatus.3 Previously, killing
the vector by aerial application of insecticides predominated as a
way of controlling such diseases. Recently, due to its comparatively
safer environmental impact, the trunk-injectable nematicides4 are
progressively replacing the conventional airborne pesticides.
Moreover, in trunk injection, it is not necessary for nematicidal
components to be highly water-soluble; hence, organic solvents,
such as ethanol, acetonitrile and acetone, can also be used.5
Although some powerful nematicidal chemicals such as milbemec-
tin (1, Fig. 1)6 and morantel tartrate7 have been used in this ap-
proach, they are expensive and ecological unsafe. Moreover, it is
feared that the nematodes are developing resistant against these
compounds;8 therefore, it is important that we discover new types
of nematicidal compounds that are safer, more effective and
economical.
in more depth later) of 5 lg/cotton ball (lg/bl.), 75 lg/bl., respec-
tively. We then set about systematically modifying these com-
pounds in order to identify molecules with greater activity. First,
regarding 3-alkylphenols, our interest focused on how the modifi-
cation of the length of alkyl group would influence the activity. In
order to investigate the structure–activity relationship (SAR), we
therefore synthesized several 3-alkylphenols, including 3. We dis-
covered 3-nonylphenol (2) showed remarkable activity (2.5 lg/
bl.).12 On the other hand, coumarin and its many derivatives are
chiefly isolated from higher plants, especially citrus fruits, and
have been found to exhibit various biological, pharmacological
activities, for example, anti-bacterial, anti-inflammatory and anal-
gesic properties.13 Until now, however, study of their nematicidal
activities has not been performed. In a pilot study, we investigated
Our previous paper proposed a convenient screening method
for nematicidal activity against B. xylophilus.9 We found by this
method that 3-undecylphenol (3) from Knema hookeriana, a Suma-
tran rainforest plant10 and coumarin (4) from Ageratum conyzoides,
a native of the South American rainforest (Fig. 1),11 both had pro-
nounced activity with a minimum effective dose (MED, discussed
* Corresponding author. Tel.: +81 48 467 9389; fax: +81 48 467 9379.
E-mail address: ktakaishi@riken.jp (K. Takaishi).
Figure 1. Structures of nematicidal compounds 1–4.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.102

K. Takaishi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5614–5617
5615
the activity of several simple coumarin derivatives, for example,
hydroxycoumarins, methylcoumarins, chlorocoumarins and 4-
methoxycoumarin (8) and 7-methoxycoumarin (14). We found
ane to 6. 4-Methoxycoumarin (8) and 7-methoxycoumarin (14)
were prepared by methylation with diazomethane of 4-hydroxy-
coumarin (7) and 7-hydroxycoumarin (13), respectively, by stan-
dard methods. 5-Methoxycoumarin (10), 6-methoxycoumarin
(12) and 8-methoxycoumarin (16) were synthesized by the Wittig
reaction of methoxy group substituted 2-hydroxybenzaldehydes
with the Wittig reagent, ethyl (triphenylphosphoranylidene)ace-
tate in N,N-diethylaniline under reflux.15 This method afforded
the desired methoxycoumarins in high yield, though Kitamura
et al. report that these coumarins were synthesized using
K2PtCl4/AgOTf in low yield.16 Commonly the stereoselectivity of
the product in the Wittig reaction depends strongly on stability
of ylides. Ethyl (triphenylphosphoranylidene)acetate is stabilized
ylides, so (E)-alkenes are generated as intermediates. In our case,
however, the desired compounds are (Z)-alkenes. Both the height
of N,N-diethylaniline’s boiling point (217 °C at 1 atm) and the sta-
bility of d-lactone contribute to isomerization of (Z)-alkenes from
(E)-alkenes. 5-Alkoxycoumarins (18–24) were prepared in two
steps from 10. In the first step, 5-hydroxycoumarin (17) was ob-
tained by demethylation of 10 with tribromoborane; in the second
step, the desired 18–24 were prepared by coupling 17 with the
appropriate bromides.
that 14 showed higher activity (MED = 40 lg/bl.) than coumarin,
whereas 8 and other related compounds lost the activity. There-
fore, we were interested in investigating the SAR, especially the ef-
fects of the methoxy group position and the length of the alkoxy
group carbon chains. Most of the variant compounds are not com-
mercially available, however, we synthesized the mono-
methoxycoumarins and their analogs. We planned to investigate
their nematicidal activities as well as toxicity to fish and crusta-
ceans, as an ecologic safety assessment index.
Scheme
1 illustrates the synthetic routes to all mono-
methoxycoumarins, 5-alkoxycoumarins and their analogs. 3-Meth-
oxycoumarin (6) was synthesized by a series of two reactions,
hydrolysis and methylation, namely 3-acetamidocoumarin (4)
was hydrolyzed with methanolic 3 N hydrochloric acid,14 yielding
3-hydroxycoumarin (5), which was methylated with diazometh-
We tested the synthesized coumarins, two alkylphenols and mil-
bemectin for three kinds of biological activities; nematicidal, toxic-
ity to crustaceans and toxicity to fish. The nematicidal assay
previously reported was used with some modifications. A cotton
ball (5 mm in diameter) containing a certain test concentration or
corresponding equivalent solvent as the control was dried in
vacuum and placed on a Petri dish (4 cm diameter) containing full
grown mycelia of Botrytis cinerea, which had been cultured on
Czapek-Dox agar medium at 21 °C for 4 days. Hundred microliters
of a distilled-water suspension of nematodes (B. xylophilus, ca.
15,000/ml) was injected into the cotton ball, and the Petri dish
was kept at 26 °C for 4 days. The minimum effective dose (MED)
was determined by observing whether the mycelia were consumed
by nematodes or not, and is denoted by the sign À (completely con-
sumed), (partly consumed) or + (not consumed).17 The highest
dose of coumarins was 160 lg/bl., and the assay was performed to
obtain MED values by the serial dilution method. The values are
each defined as the lowest dose of the test compound to show activ-
ity ( ). If the mycelia are not consumed, it is conceivable that the
tested compound has a temporary antifeedant or paralytic activity,
as opposed to genuine nematicidal activity. Therefore, nematodes
were promptly recovered about those in which active was observed
after evaluation, survival was evaluated by methylene blue staining,
and nematicidal activity was confirmed in all instances. Crustacean
lethality was tested using a brine shrimp, Artemia salina, which is
widely used for experiments on the safety of agricultural chemi-
cals,18 as follows: the eggs of A. salina were placed in seawater; they
hatched within 48 h to provide large number of larvae for experi-
mental use. Compounds dissolved in dimethyl sulfoxide (50 ll)
were tested in vials containing 5 ml of seawater and ten shrimps
in each of three replicates. Survivors were counted after 24 h, and
LC100 and LC50 were determined. The evaluation system that con-
tained only dimethyl sulfoxide was used as a control in this case.
A test of fish toxicity was done using Japanese killifish, Oryzias lati-
pes, fasted for 24 h before the test. O. latipes is a freshwater fish
which, like A. salina, is commonly used for evaluation the safety of
compounds.19 Tested compounds were dissolved in acetone
(1.0 ml) and added to beakers containing 150 ml of demineralized
oxygen-rich water and five fish in each of three replicates. Survivors
were counted after 24 h and judged LC100 and LC50. The evaluation
system that contained only acetone was used as a control in this
case. The highest concentration of test compounds was 80 ppm in
the A. salina lethality test, 100 ppm in the O. latipes lethality test;
both assays were performed by the serial dilution method.
Scheme 1. Reagent and conditions: (a) 3 N methanolic HCl, reflux, 99%; (b) CH2N2,
Et2O, rt (35% for 6, 23% for 8, 25% for 14); (c) Ph3P = CHCO2Et, N,N-diethylaniline,
reflux (71% for 10, 98% for 12, 82% for 16); (d) BBr3, CH2Cl2, À78 °C, 84%; (e) RBr,
K2CO3, acetone, rt (quant. for 18, 92% for 19, 90% for 20, 97% for 21, 83% for 22, 88%
for 23, 83% for 24).

5616
K. Takaishi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5614–5617
Table 1
Nematicidal activity of coumarin derivatives, and toxicity against crustacea and fish.
Compound
Substituent site-group of coumarin
Nematicidal activitya (MED) (
lg/bl.)
Lethal effect on A. salina (ppm)
Lethal effect on O. latipes (ppm)
LC100
LC50
Selectivity indexb
LC100
LC50
Selectivity indexb
1
2
3
4
6
8
10
12
14
16
25
18
19
20
21
22
23
24
17
26
0.6
2.5
5.0
80
80
nac
20
80
40
80
20
10
20
40
40
40
80
nac
nac
nac
0.6
1.3
1.3
>80
>80
>80
20
>80
80
>80
0.1
0.6
1.0
>80
>80
60
30
>80
20
0.17
0.24
0.20
>1.00
>1.00
—
1.50
>1.00
0.50
>1.00
0.25
10
5
0.06
7.5
3.8
38
>100
75
38
75
50
100
0.10
3.01
7.06
0.48
>1.25
—
1.90
0.94
1.25
1.25
Nothing
3-OMe
4-OMe
5-OMe
6-OMe
7-OMe
8-OMe
5,7-Di-OMe
5-OEt
50
>100
100
50
100
75
>80
>100
>80
>80
>8.00
100
75
7.52
5-OPr
5-OBu
5-O-Pentyl
5-O-Octyl
5-OBn
5-O-Allyl
5-OH
a
Maximum dose of the test sample is 160 lg/bl.
(1/MED)/(1/LC50).
No activity.
b
c
as the 1/MED (lg/bl.) divided by 1/LC50 (ppm). High values of this
metric reflect a high degree of selectivity for nematodes. The selec-
tivity index values of 1, taken as a benchmark for that of other
compounds were 0.17 (against A. salina) and 0.10 (against O. lati-
pes). Meanwhile, the selectivity values of the coumarins were
much higher than those of 1–3. In particular, 18 was 75 (or more)
times more selective than 1.
Figure 2. Structures of 25 and 26.
Systematic evaluation of coumarin derivatives for nematicidal
activity has revealed a SAR, with activity associated with methoxy
group and 5-alkoxy groups. We have discovered that 5-ethoxy-
coumarin shows high nematicidal activity. Both A. salina and O. lat-
ipes lethality tests suggested that this compound is much safer
than milbemectin and alkylphenols. From this work, we conclude
that 5-ethoxycoumarin is comparatively potent and it should be
considered a lead compound for the development of new class of
the nematocides.
The result of the bioassay is shown in Table 1. Among the cou-
marin derivatives, behaviors depend strongly on the position and
the type of substituent. Among the monomethoxycoumarins, 10
showed the highest nematicidal activity (MED = 20
activity of 14 was comparatively high (MED = 40
l
g/bl.). The
l
g/bl.) while
the other monomethoxycoumarins did not have high activities.
5,7-Dimethoxycoumarin (25, Fig. 2), bears both substitution found
in 10 and 14, and was predicted to have higher activity; however,
the compound was comparable to 10. Therefore, we planned to
investigate the nematicidal activity of alkoxycoumarins and their
analogs with a focus on the 5-position; prior to this study, the bio-
activity of these compounds had been hardly studied. Among the
5-alkoxycoumarins, 5-ethoxycoumarin (18) showed a higher activ-
Acknowledgments
We thank Dr. Keiji Tanaka of Sankyo Agro Co., Ltd (the current
affiliation is Daiichi Sankyo Co., Ltd), for furnishing us with milbe-
mectin. We also thank the SC-NMR Laboratory of Okayama Univer-
sity for 600, 500 and 300 MHz NMR experiments and the MS
Laboratory of the Faculty of Agriculture of Okayama University.
ity (MED = 10 lg/bl.) than 10. The activities of 17, 5-benzyloxy-
coumarin (23) and 5-allyloxycoumarin (24) were much lower
than 18; leading us to conclude that in addition to position and
the length of the alkoxy group, saturation grade is also important.
Moreover, we confirmed that (E)-coumarinic acid (26, Fig. 2) ((Z)-
26 is unstable), the open form of coumarin, did not show nemati-
cidal activity. In the both the A. salina and O. latipes lethality tests of
the main compounds, 10 and 14 showed higher toxicity than other
coumarins, in agreement with their increased nematicidal activity.
The toxicity of 18, however, was low against both A. salina and O.
latipes, though it showed high nematicidal activity. On the other
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.08.102.
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